Biochip and target dna quantitative method

ABSTRACT

A biochip used for quantitative analysis of a target DNA contained in a sample, includes a type I chamber that includes: a primer that is designed to bind to the target DNA; a internal standard DNA of a first amount that has a sequence different from a sequence of the target DNA, and is amplifiable with the primer; and a fluorescent probe that is designed to bind to a part of an PCR product of the target DNA and to a part of an PCR product of the internal standard DNA, and fluoresces differently for the PCR product of the target DNA and the PCR product of the internal standard DNA, and a type II chamber including: the internal standard DNA of a second amount different from the first amount; and the primer and the fluorescent probe.

CROSS-REFERENCE

This application claims priority to Japanese Patent Application No.2009-214712, filed on Sep. 16, 2009, the entirety of which is herebyincorporated by reference.

BACKGROUND

1. Technical Field

The present invention relates to biochips and target DNA quantitativemethods used for quantitative analysis of target DNAs.

2. Related Art

Methods of chemical analysis, chemical synthesis, and other proceduresincluding various analyses in bio-related fields using a microfluidicchip that includes microchannels in a glass substrate or the like havegained attention. The microfluidic chip also has other names, such as amicro total analytical system (micro TAS) and a lab-on-a-chip. Becauseof advantages such as smaller amounts of samples and reagents, shorterreaction time, and less waste over common analyzing devices, themicrofluidic chip is expected to have a wide range of applications,including medical diagnoses, on-site analyses of environment and food,and production of drugs and chemicals. Because the microfluidic chiprequires small amounts of reagents, the amounts of expensive reagentscan be reduced, and thus the cost of tests can be reduced. Requiringsmall amounts of samples and reagents means a shorter reaction time andimproved test efficiency. Because samples such as blood are used insmall amounts, the use of the microfluidic chip for medical diagnosesadvantageously reduces the burden on patients.

The PCR (Polymerase Chain Reaction) method is well known as the methodof amplifying nucleic acids, including DNA and RNA. In the PCR method, amixture of target DNA and reagents is put into a tube, and a reaction isallowed in several minutes of repeated cycles at three differenttemperatures, for example, 55° C., 72° C., and 94° C., using atemperature control device called a thermal cycler. The enzyme (DNApolymerase) acts only on the target DNA and amplifies it about two-foldin each temperature cycle.

Real-time PCR, a technique that uses specific fluorescent probes toquantity DNA as it is amplified, has been put to practical applications.Because of high measurement sensitivity and reliability, real-time PCRhas been widely used in research and clinical tests.

In real-time PCR, however, the amplification inhibitors present in asample may lower the reliability of the results.

Simultaneous measurements of large numbers of reaction systems areusually difficult, because the amounts of DNA extracted from a sampleare generally small, and the reagents used for the PCR are usuallyexpensive. The amount of reaction mixture required for PCR is typicallyseveral ten microliters, and basically only a single gene can be assayedper reaction system.

WO/2005/059548 describes a method for assaying nucleic acids with theuse of a fluorescent probe that hybridizes with both the target DNA andan internal standard DNA. Because this method coamplifies the target DNAand the internal standard DNA, the influence of amplification inhibitorsis small, and an expensive, dedicated real-time PCR apparatus is notnecessary. However, the method greatly suffers from poor quantificationaccuracy, particularly when there is a large concentration differencebetween the target DNA and the internal standard DNA, and thequantifiable concentration range of the target DNA is small.

SUMMARY

An advantage of some aspects of the invention is to provide a biochipand a target DNA quantitative method with which the target DNA containedin a biological sample can be quantified under the small influence ofamplification inhibitors contained in the sample, and which allow for anucleic acid amplification reaction and the quantification of the targetDNA with ease over a wide concentration range of the target DNA.

To achieve the above stated advantage, the present invention provides abiochip used for quantitative analysis of a target DNA contained in asample. The biochip comprises: a type I chamber includes; a primer thatis designed to bind to the target DNA; a internal standard DNA of afirst amount that has a sequence different from a sequence of the targetDNA, and is amplifiable with the primer; and a fluorescent probe that isdesigned to bind to apart of a PCR product of the target DNA and to apart of a PCR product of the internal standard DNA, and fluorescesdifferently for the PCR product of the target DNA and the PCR product ofthe internal standard DNA, and a type II chamber includes; the internalstandard DNA of a second amount different from the first amount; and theprimer; and the fluorescent probe.

The biochip may further comprise; a first path connected to the type Ichamber and the type II chamber; and a reservoir connected to the firstpath.

The biochip may further comprise; a first group of chambers includes;the type I chamber and the type II chamber that include a first primerused to amplify a first target DNA, and a second group of chambersincludes; the type I chamber and the type II chamber that include asecond primer different from the first primer and used to amplify asecond target DNA different from the first target DNA.

The present invention also provides a quantitative method for a targetDNA contained in a biological sample using a biochip. This methodcomprises: introducing a sample into a type I chamber and a type IIchamber of the biochip; the type I chamber includes; a primer that isdesigned to bind to the target DNA; a internal standard DNA of a firstamount that has a sequence different from a sequence of the target DNA,and is amplifiable with the primer; and a fluorescent probe that isdesigned to bind to a part of a PCR product of the target DNA and to apart of a PCR product of the internal standard DNA, and fluorescesdifferently for the PCR product of the target DNA and the PCR product ofthe internal standard DNA, and the type II chamber includes; theinternal standard DNA of a second amount different from the firstamount; the primer; and the fluorescent probe, performing a PCRamplification in the type I chamber and the type II chamber; measuring afluorescence intensity of the fluorescent probe in the type I chamberand the type II chamber; determining a regression curve of equation (1)below based on the measured fluorescence intensities in the type Ichamber and the type II chamber, and amounts of the internal standardDNA contained in the type I chamber and the type II chamber,

F=a/(C+b)+c  (1)

where F is the fluorescence intensity, C is an amount of internalstandard DNA, b is an amount of target DNA contained in the sample, anda and c are predetermined values; and estimating the amount of thetarget DNA contained in the sample based on the regression curve.

In the biological sample quantification method, the regression curve maybe determined based on a relationship between a ratio of fluorescenceintensities measured before and after the PCR amplification reaction andthe amount of the internal standard DNA used in the type I chamber andthe type II chamber.

In the biological sample quantification method, the regression curve maybe determined based on a relationship between the ratio of afluorescence intensity measured in a first state in which thefluorescent probe binds to a part of the PCR product after the PCRamplification, and a fluorescence intensity measured in a second statein which the fluorescent probe dissociates from the PCR product afterthe PCR amplification and the amount of the internal standard DNA usedin the type I chamber and the type II chamber.

In the biochip, the type I chamber and the type II chamber include knownamounts of internal standard DNA, a primer, and a fluorescent probe. Theprimer is designed to bind to the target DNA. The internal standard DNAhas a sequence different from a sequence of the target DNA, and isamplifiable with the primer. The fluorescent probe is designed to bindto a part of a PCR product of the target DNA and to a part of a PCRproduct of the internal standard DNA. The fluorescent probe fluorescesdifferently upon binding to the PCR product of the target DNA and uponbinding to the PCR product of the internal standard DNA. Because a PCRamplification is performed in the type I chamber and the type II chamberthat contain different amounts of internal standard DNA in each other, auser is able to easily perform a PCR amplification and thequantification of the target DNA simply by introducing a sample into thetype I chamber and the type II chamber. The quantification of the targetDNA using the biochip can be performed under the small influence of theamplification inhibitors that may be present in the sample, and over awide quantifiable range of the target DNA. Because the introduction of asample into the biochip is all that is required, a user does not need togo through laborious procedures. Because only small amounts of reagentsare used, the biochip enables quantification of the target DNA at lowcost and with high accuracy.

The biological sample quantification method is a method for quantifyingthe target DNA contained in a sample using the biochip. The methodincludes determining a regression curve of equation (1) above based onthe measured fluorescence intensity in the type I chamber and the typeII chamber, and the amount of internal standard DNA in the type Ichamber and the type II chamber, and estimating the amount of the targetDNA contained in the sample based on the regression curve. Thus, the DNAamplification is performed under the small influence of theamplification inhibitors that may be present in the sample, and over awide quantifiable range of the target DNA. Because the introduction of asample into the biochip is all that is required, a user does not need togo through laborious procedures. Because only small amounts of reagentsare used, the quantification of the target DNA can be performed at lowcost and high accuracy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a plan view schematically illustrating a configuration of amicrochamber array according to an embodiment of the invention; FIG. 1Bis a cross sectional view at C-C of FIG. 1A.

FIG. 2 is a side view of a centrifugal device in which the microchamberarray according to an embodiment of the invention is installed.

FIG. 3 is a plan view of the centrifugal device of FIG. 2 as viewed fromabove.

FIGS. 4A, 4B, and 4C are views illustrating the microchamber arrayattached to a holder of the centrifugal device of FIG. 2, as viewed fromabove.

FIGS. 5A, 5B, and 5C are transverse sectional views of the microchamberarray attached to the holder of the centrifugal device of FIG. 2.

FIG. 6 is a view showing examples of the sequences of a target DNA and ainternal standard DNA used in Example 1 of the invention.

FIG. 7 is a view showing an example of the sequences of primers used inExample 1 of the invention.

FIG. 8 is a view showing a sequence of a Q-probe used in Example 1 ofthe invention.

FIG. 9 is a diagram representing the relationship (regression curve)between the amount of internal standard DNA (log C) and fluorescenceintensity (amount of change) F in the samples quantified in Example 1 ofthe invention.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

Preferred embodiments of the present invention will be described belowwith reference to the drawings.

1. Embodiment 1.1. Biochip

FIG. 1A is a plan view schematically illustrating a configuration of amicrochamber array (biochip) 10 according to an embodiment of theinvention. FIG. 1B is a cross sectional view at C-C of FIG. 1A.

The microchamber array 10 is a biochip (biological sample quantificationchip) used to quantify the target DNA contained in a sample. Asillustrated in FIG. 1A and FIG. 1B, the microchamber array 10 includesgroups of chambers 201, 202, 203, 204, 205, and 206 that contain aplurality of chambers 104. The microchamber array 10 also includes firstpaths 105 connected to each chamber 104. A reservoir 107 and a wastestorage 106 connected to the first paths 105 are also provided. Thewaste storage 106 is connected to the reservoir 107 via the first paths105. The microchamber array 10 further includes second paths 108 thatconnect the first paths 105 to the waste storage 106, and a inlet 109through which a reaction liquid is externally supplied into thereservoir 107 of the microchamber array 10.

As illustrated in FIG. 1B, the microchamber array 10 is made bycombining transparent substrates 101,102, and 103. The transparentsubstrate 101 includes the chambers 104, the first paths 105, thereservoir 107, and the inlet 109. The transparent substrate 102 includesthe waste storage 106 and the second paths 108. The transparentsubstrates 101, 102, and 103 are, for example, resin substrates. Forexample, injection molding can be used to form the chambers 104, thefirst paths 105, the reservoir 107, the inlet 109, the waste storage106, and the second paths 108.

The chambers 104 hold known amounts of internal standard DNA, and aprimer and a fluorescent probe. The internal standard DNA has a sequencedifferent from the sequence of the target DNA, and is amplifiable withthe primer common to the target DNA to be quantified. The primer isdesigned to bind to the target DNA and to the internal standard DNA. Thefluorescent probe is designed to bind to a part of the PCR product ofthe target DNA, and to a part of the PCR product of the internalstandard DNA, and fluoresces differently upon binding to the PCR productof the target DNA and upon binding to the PCR product of the internalstandard DNA. More specifically, the internal standard DNA, the primer,and the fluorescent probe are placed on the surfaces of the chambers 104by applying to the surfaces of the chambers 104 followed by drying. Inthe microchamber array 10, the chambers 104 that belong to one group ofchambers contain different amounts of internal standard DNA. That is,one group of chambers comprises a type I chamber that includes a firstamount of a internal standard DNA, and a type II chamber that includes asecond amount of the internal standard DNA.

As noted above, the microchamber array 10 includes the groups ofchambers 201, 202, 203, 204, 205, and 206. The chambers 104 in one groupof chambers contain the same internal standard DNA, the same primer, andthe same fluorescent probe. A column of chambers (chambers 104 in acolumn) in the microchamber array 10 may contain the same amount ofinternal standard DNA. Here and below, in the microchamber array 10, theseries of chambers 104 in a group of chambers is a row, and the seriesof chambers in a direction perpendicular to the rows are columns A to F.

The chambers 104 contained in a first group of chambers may include afirst primer used to amplify a first target DNA, and the chambers 104contained in a second group of chambers may contain a second primer usedto amplify a second target DNA different from the first target DNA. Inthis way, the microchamber array 10 can be used to quantify differenttarget DNAs.

The chambers 104 in the first group of chambers contain a firstfluorescent probe that changes a fluorescence amount upon binding to thefirst target DNA amplified in the chambers 104 in the first group ofchambers. The fluorescent probe is designed to change a fluorescenceamount also upon binding to the internal standard DNA of the firsttarget DNA. The amount of fluorescence change of the fluorescent probeis different when it binds to the first target DNA and to the internalstandard DNA.

The chambers 104 in the second group of chambers contain a secondfluorescent probe that changes a fluorescence amount upon binding to thesecond target DNA and upon binding to the internal standard DNA of thesecond target DNA. The second target DNA has different sequence from thesequence of the first target DNA amplified in the chambers 104 containedin the first group of chambers. In this way, the microchamber array 10can be used to calculate regression curves of more than one target DNA.

The chambers 104 are, for example, cylindrical in shape, and have across sectional diameter of 500 μm, and a depth of 100 μm. The firstpaths 105 have a width of 200 μm and a depth of 100 μm in the crosssection perpendicular to the direction of flow of the reaction mixture.The each 2 chambers 104 are spaced apart by a distance sufficient toprevent the reaction mixture from being mixed between the chambers 104.

Preferably, the inner surfaces of the chambers 104 and the first paths105 are rendered lyophilic by a surface treatment to prevent adsorptionof air bubbles. Preferably, the inner surfaces of the chambers 104 andthe first paths 105 are subjected to a surface treatment that suppressesnon-specific adsorption of biomolecules such as proteins.

The waste storage 106 is connected to the first paths 105 via the secondpaths 108. As will be described later, the waste storage 106 has alarger volume than that of the first paths 105, because the reactionmixture filling the first paths 105 discharges into the waste storage106. The second paths 108 are formed perpendicularly through thetransparent substrate 102 (for example, φ=90° in FIG. 1B).

The contact faces of the transparent substrates 101, 102, and 103 may besubjected to a liquid-repelling surface treatment, or may have a sealingproperty to prevent the reaction mixture from leaking out of thechambers 104 and entering other chambers 104 through the contact facesof the substrates. For example, leaking of the reaction liquid from thechambers 104 can be prevented by coating the contact faces with siliconerubber or fluororesin.

1.2. Biological Sample Quantification Method

The target DNA quantitative method according to an embodiment of theinvention is a method for quantifying the target DNA contained in abiological sample using the microchamber array (biochip) 10 of thepresently described embodiment. The method includes introducing a sampleinto each chamber 104; performing a PCR amplification in the chambers104; measuring the fluorescence intensity of the fluorescent probe ineach chamber 104, which bound to a part of the amplified DNA;determining a regression curve of the equation (1) below based on themeasured fluorescence intensity in each chamber 104, and the amount ofinternal standard DNA used in each chamber 104; and estimating theamount of target DNA contained in the sample based on the regressioncurve.

F=a/(C+b)+c  (1),

where F is the fluorescence intensity, C is the amount of internalstandard DNA, b is the amount of target DNA contained in the sample, anda and c are predetermined values.1.2.1. Introduction of Sample into the Chambers (Reaction MixtureFilling Method)

The following describes how a sample is introduced into the chambers 104of the microchamber array 10. In the present embodiment, a method offilling the chambers 104 with the reaction mixture prepared from asample is described. First, a user supplies the reaction mixture to thereservoir 107 through the inlet 109 using a pipette or the like.

The reaction mixture is prepared from a sample. For example, thereaction mixture contains the target DNA, DNA polymerase, andnucleotides (dNTPs) of concentrations suited for reaction.

The target DNA is DNA extracted from biological samples such as blood,urine, saliva, and spinal fluid, or cDNA reverse-transcribed from theextracted RNA.

The user then rotates the microchamber array 10 with a centrifugaldevice 50 illustrated in FIG. 2 and FIG. 3. FIG. 2 is a side view of thecentrifugal device 50. FIG. 3 is a plan view of the centrifugal device50 as viewed from above.

As illustrated in FIG. 2 and FIG. 3, the centrifugal device 50 includesholders (rotated members) 51 attachable to the microchamber array 10,and a rotary motor (rotating member) 52. Each holder 51 is tilted withangle θ made by the microchamber array 10 with respect to the rotationalaxis o. Accordingly, the microchamber array 10 attached to the holder 51is also tilted with angle θ made by the microchamber array 10 withrespect to the rotational axis o. Here, θ=45°. However, the angle θ isnot limited as long as it falls within the range of 0°<θ<90°.

FIGS. 4A, 4B, and 4C are plan views of the microchamber array 10attached to the holder 51 of the centrifugal device 50, as viewed fromabove. FIGS. 5A, 5B, and 5C are transverse sectional views of themicrochamber array 10 attached to the holder 51. FIGS. 5A, 5B, and 5Care D-D cross sections of FIGS. 4A, 4B, and 4C, respectively.

As illustrated in FIG. 4A and FIG. 5A, the user operates the centrifugaldevice 50 after setting the microchamber array 10 on the holder 51 insuch a manner that the transparent substrate 101 is on the outer sidewith respect to the rotational axis o. The rotation exerts a centrifugalforce in a direction from the reservoir 107 to the chambers 104, causingthe reaction mixture in the reservoir 107 to move along the first paths105 and fill the chambers 104. Air, having a lower specific gravity thanthe reaction mixture, is pushed out from chambers 104 into the firstpaths 105. The reaction mixture fills the chambers 104 as it replacesthe air.

Here, the reaction mixture is not sent into the waste storage 106. Thisis because, as illustrated in FIG. 5A, the second paths 108 directedfrom the first paths 105 to the waste storage 106 makes a 135° anglewith respect to the direction of the centrifugal force (the direction ofarrow F), and thus the centrifugal force that acts in the direction fromthe first paths 105 to the waste storage 106 is 0 or less.

The reaction mixture is not sent into the waste storage 106 as long asthe angle made by the direction of the centrifugal force and the secondpaths 108 directed from the first paths 105 to the waste storage 106 is90° or more and 180° or less. Thus, when θ=45°, the reaction liquid isnot sent into the waste storage 106, provided that the angle φ made bythe transparent substrate 102 and the second paths 108 as in FIG. 1Bfalls within the range of 45°<φ≦135°.

As described above, because the reaction mixture does not flow towardsthe waste storage 106, the reaction mixture can efficiently fill all thechambers 104. After the rotation, as illustrated in FIG. 4B and FIG. 5B,all the chambers 104 and the first paths 105 are filled with thereaction mixture.

The user then stops the rotation of the centrifugal device 50, and, asillustrated in FIG. 4C and FIG. 5C, resumes the operation of thecentrifugal device 50 after attaching the microchamber array 10 to theholder 51 in such a manner that the transparent substrate 103 is on theouter side with respect to the rotational axis o. The reaction mixturein the first paths 105 is sent into the waste storage 106. This isbecause, as illustrated in FIG. 5C, the second paths s 108 directed fromthe first paths 105 to the waste storage 106 makes a 45° angle withrespect to the direction of the centrifugal force (the direction ofarrow F), and thus the centrifugal force in the direction from the firstpaths 105 to the waste storage 106 is 0 or more.

The reaction mixture is sent into the waste storage 106 as long as theangle made by the direction of the centrifugal force and the secondpaths 108 directed from the first paths 105 to the waste storage 106 is0° or more and less than 90°. Thus, when θ=45°, the reaction mixture issent into the waste storage 106, provided that the angle φ made by thetransparent substrate 102 and the second paths 108 as in FIG. 1B fallswithin the range of 45°<φ≦135°. The reaction mixture in the first paths105 is sent into the waste storage 106, whereas the reaction mixture inthe chambers 104 remains therein.

The chambers 104 are isolated from each other after the reaction mixturein the first paths 105 has been sent into the waste storage 106.

When the microchamber array 10 is set on the holder 51 for rotation inthe state illustrated in FIG. 4C and FIG. 5C, the user may supply amineral oil to the reservoir 107 through the inlet 109 using a pipetteor the like. In this case, the first paths 105 are filled with themineral oil upon rotation of the microchamber array 10. The reactionmixture in the chambers 104 is not replaced with the mineral oil becausethe reaction mixture has a higher specific gravity than that of themineral oil. The isolation of each chamber 104 with the use of the oilprevents contamination between the chambers 104 (mixing of the reactionmixture in one chamber with others). Drying of the chambers 104 duringthe reaction process also can be prevented. A liquid having a lowerspecific gravity than that of the reaction liquid, and that isimmiscible with the reaction mixture and does not evaporate as easily asthe reaction mixture may be used instead of the mineral oil. The usermay rotate the centrifugal device 50 in the state illustrated in FIG. 4Cand FIG. 5C to send the reaction mixture in the first paths 105 to thewaste storage 106, and may resume the rotation of the centrifugal device50 after supplying mineral oil to the reservoir 107.

PCR (biological sample reaction) is performed after the reaction mixturehas been supplied to the microchamber array 10 according to theprocedures described above. Specifically, the microchamber array 10 isinstalled in a thermal cycler for PCR after sealing the opening of themicrochamber array 10. Generally, cycling steps of dissociatingdouble-stranded DNA at 94° C., annealing the primer at about 55° C., andreplicating the complementary strands at about 72° with a heat-stableDNA polymerase are repeated.

After the PCR, the fluorescence intensity in each chamber 104 ismeasured with a fluorescence microscope to quantify the amount of targetDNA contained in the reaction mixture in each chamber 104.

As described above, the microchamber array 10 of the present embodimentutilizes a centrifugal force to supply the reaction mixture into thechambers 104 through the first paths 105, and thus enables a reactionprocess for minute quantities of reaction mixture that cannot be easilyquantified with the use of a pipette. Because the reaction mixture canbe processed in large numbers of chambers 104 at once, different testsor procedures can be efficiently performed.

The centrifugal force is applied to first fill the first paths 105 andthe chambers 104 with the reaction mixture, and reapplied in a differentdirection to send out the reaction mixture in the first paths 105 to thewaste storage 106. In this way, contamination between the chambers 104can be prevented, because the reaction is performed in isolation in eachchamber 104. The present embodiment has been described through the useof a centrifugal force to fill the chambers 104 with the reactionmixture. However, other forces, such as the pressure of a capillaryforce or a pump may be used instead of a centrifugal force to fill thechambers 104.

The microchamber array 10, used as a reaction apparatus for PCR in thisembodiment, also can be used for other DNA amplification reactions (forexample, LAMP method).

1.2.2. Quantification of Target DNA

The target DNA quantitative method of the present embodiment furtherincludes performing a PCR amplification in the chambers 104; measuringin each chamber 104 the fluorescence intensity of the fluorescent probebound to a part of the amplified DNA; determining a regression curve ofthe equation (1) above based on the measured fluorescence intensity ineach chamber 104, and the amount of internal standard DNA used in eachchamber 104; and estimating the amount of target DNA contained in thesample based on the regression curve.

As described above, the chambers 104 include known amounts of internalstandard DNA, and a primer and a fluorescent probe. Thus, in the PCRamplification in the chambers 104, the internal standard DNA placed inadvance in the chambers 104, and the target DNA contained in thereaction mixture introduced into the chambers 104 are both amplified inthe chambers 104.

The PCR amplification may be such that, for example, the fluorescentprobe is designed to bind to the target DNA and to the internal standardDNA, and that the fluorescence of the fluorescent probe is quenched uponbinding to the target DNA or the internal standard DNA. Fluorescenceintensity in the chambers 104 is measured in this state. A regressioncurve of the equation (1) above is then determined based on the measuredfluorescence intensities in the chambers 104, and the amount of internalstandard DNA used in each chamber 104. The regression curve can then beused to estimate the amount of the target DNA contained in the sample.

The fluorescence intensity (F) in equation (1) may be (i) thefluorescence intensity of the fluorescent probe, (ii) the ratio offluorescence intensities that is measured before the PCR amplificationand after the PCR amplification, or (iii) the ratio of the fluorescenceintensity measured in a heated temperature state in which the PCRproduct and the fluorescent probe dissociate from each other (a firststate in which the fluorescent probe dissociates from the amplified DNAafter the PCR amplification), and the fluorescence intensity measured atthe binding temperature of the fluorescent probe (a second state inwhich the fluorescent probe binds to a part of the PCR product after thePCR amplification).

Assume here that the amount of fluorescence change when all the PCRproducts of the reaction are the PCR products of target DNA (X) is Ft,and that the amount of fluorescence change when all the PCR products arethe PCR products of internal standard DNA (C) is Fc. When the chambers104 include both the internal standard DNA and the target DNA, the PCRamplification produces the PCR products of both the internal standardDNA and the target DNA. The amount of fluorescence change F for thesePCR products can be given by the following equation (2).

F=FtX/(X+C)+FcC/(X+C)=[X(Ft−Fc)/(X+C)]+Fc  (2),

where C is the amount of internal standard DNA in the chambers 104 (copynumber), and X is the amount of target DNA in the chambers 104 (copynumber).

Equation (1) can be derived from equation (2). A regression curve ofequation (1) can be obtained by plotting the amount of fluorescencechange (F) of each chamber 104 on the vertical axis against the amountof internal standard DNA (log C) contained in each chambers 104 of thesame group of chambers on the horizontal axis (see, for example, FIG. 9described later). The value of b in the three parameters a, b, and c inequation (1) determined from the regression curve corresponds to theamount of the target DNA.

Any fluorescent probe can be used as long as it can bind to a part ofthe PCR product of the target DNA, and shows different fluorescencechanges for the target DNA and the internal standard DNA. Examples ofsuch fluorescent probes include Taqman probe®, Hyb probe®, MolecularBeacon®, and Q-Probe®. Q-Probe is a probe used to detect target DNAs byutilizing the “fluorescence quenching phenomenon”, in which theintensity of emitted fluorescence decreases as a guanine base approachesa labeled fluorescent dye.

Q-Probe is designed to have a sequence that has a fluorescent-labeledcytosine at one terminal, and that specifically binds to the target DNA.Upon binding of the Q-Probe to the target DNA, fluorescence intensitydecreases under the influence of a guanine. When the target DNAquantitative method of the present embodiment is to use a competitivePCR method with a Q-Probe, the base of the target DNA corresponding tothe fluorescent-labeled terminal base of the Q-probe cannot be a guaninewhen a guanine is the corresponding base of the internal standard DNAfor the fluorescent-labeled terminal base of the Q-Probe. As long asthis is the case in the coamplification of the target DNA and theinternal standard DNA using a Q-probe, the fluorescence intensity of thelabeled fluorescent dye decreases (quenches) upon hybridization of theinternal standard DNA with the Q-Probe, whereas the fluorescenceintensity of the labeled fluorescent dye does not decrease uponhybridization of the target DNA with the Q-Probe. Here, the target DNAand the internal standard DNA can be read the other way around. That is,whether to cause quenching of the fluorescence upon binding of theQ-Probe to the target DNA or the internal standard DNA is arbitrary.

The target DNA quantitative method of the present embodiment can be usedto quantify multiple test items from a sample, using the groups ofchambers of the microchamber array 10. For example, the method can beused for the gene testing of food poisoning-causing microorganisms(specifically, for the testing of pathogenic microorganisms in food, orthe testing of pathogenic microorganisms in a sample (feces) collectedfrom patients suffering from food poisoning).

In this case, genes of the pathogenic microorganisms are used as thetarget DNAs in the chambers 104 of the groups of chambers, and theinternal standard DNAs set for these target DNAs are placed in thesegroups of chambers. Genes of different causative microorganisms can bequantified in each group of chambers. Examples of food poisoning-causingmicroorganisms include campylobacters, salmonellas, Pseudomonasaeruginosa, pathogenic Escherichia coli O-157, and Staphylococcusaureus. Fluorescent probes (for example, Q-probe) designed to hybridizewith the target DNAs and the internal standard DNAs are then placed inthe chambers 104 of each group of chambers, and the target DNAs arequantified using the quantitative method described above. In this way,multiple items of target DNA quantification can be performed with easefrom a single sample.

2. Examples

The following describes the present invention in more detail based on anexample. It should be noted, however, that the invention is not limitedto the following.

This example describes a quantification method of the target DNA in asample using PCR with the microchamber array 10 of FIGS. 1A and 1B,using a Q-Probe (Kurata et al., Nucleic acids Research, 2001, vol. 29,No. 6 e34) as the fluorescent probe.

The fluorescence of the Q-Probe is quenched by the interaction with theguanine contained in the nucleic acid to which the Q-probe binds. Whenthe Q-probe is that which can bind to both the target DNA and theinternal standard DNA, and has fluorescence quenched upon binding to oneof the target DNA and the internal standard DNA, a relative amount ofthe target DNA with respect to known amounts of internal standard DNAcan be estimated.

The inventor purchased the target DNA and the internal standard DNA fromJ-Bio21 Corporation. The inventor used a mixture of 10 mM Tris-HClbuffer (pH: 8.3), 50 mM KCl, and 1.5 mM MgCl₂.

FIG. 6 represents examples of target DNA and internal standard DNAsequences. The Q-probe binds to the underlined portions. As shown inFIG. 6, the target DNA has the sequence TTTT, and the internal standardDNA the sequence GGGT immediately after the binding sites (underlinedportions) for the fluorescent probe. Thus, the fluorescence of theQ-Probe is quenched by the reaction with the guanine (G) at the bindingsite upon binding to the internal standard DNA.

The inventor placed the primers and the fluorescent probe by applying onthe surfaces of the chambers 104 of the microchamber array 10 of FIGS.1A and 1B followed by vacuum drying. The primers have the sequencesshown in FIG. 7. The fluorescent probe (Q-Probe) has the sequence shownin FIG. 8. The Q-Probe was fluorescent-labeled using a BODIPY FL(Molecular probes).

Known amounts of internal standard DNA were applied and vacuum dried onthe surfaces of the chambers 104 in different amounts for the chambercolumns A to F. Table 1 shows the amounts (copy numbers) of internalstandard DNA placed in the chambers 104 of the chamber columns A to F.

TABLE 1 Chamber column Internal standard DNA A 100 copies B 1,000 copiesC 10,000 copies D 100,000 copies E 1,000,000 copies F —

The inventor then charged a target DNA-containing reaction liquid intothe chambers 104, and conducted PCR using a thermal cycler (MasterCycler®, Eppendorf; a LightCycler 480®, Roche Diagnostics). The reactionmixture contained a LightCycler 480® Genotyping Master, and a uracil DNAglycosylase (both purchased from Roche Diagnostics). Quantificationaccuracy was ascertained by using four samples 1 to 4 with differentamounts (copy numbers) of target DNA in four microchamber arrays 10(Table 2). Fluorescence measurements were made at room temperaturebefore the amplification reaction, and at room temperature, 60° C., and95° C. after the amplification reaction.

TABLE 2 Sample Target DNA 1 100 copies 2 1,000 copies 3 10,000 copies 4100,000 copies

The chamber columns A to F contained different amounts of internalstandard DNA. FIG. 9 is a graph representing the amount of fluorescencechange (F) of each chamber 104 plotted on the vertical axis against theamount of internal standard DNA (log C) on the horizontal axis forsamples 1 to 4. Regression curves of samples 1 to 4 were obtained fromthe graph. Then, the parameters a, b, and c in equation (1) were foundfrom the regression curves. The value of b corresponds to the amount ofthe target DNA. From FIG. 9 and equation (1), the values of b (amountsof target DNA) for samples 1 to 4 were calculated as 40, 850, 9,210, and89,700 copies, respectively.

This example shows the quantification result for only one kind of targetDNA. However, the microchamber array 10 can be used for thequantification of different target DNAs by introducing different targetDNAs and reagents (primers, fluorescent probes) for the amplificationand quantification of these target DNAs to each group of chambers.

As described above, the microchamber array 10 in this example is used toconduct nucleic acid amplification reaction (PCR) using differentamounts of internal standard DNA in the chambers 104 of one group ofchambers. The fluorescence intensity in each chamber 104 can be obtainedby measuring the fluorescence intensity of the fluorescent probe thathas bound to a part of the PCR amplified DNA. A regression curve ofequation (1) expressed as the relationship between the amount offluorescence change (F) and the amount of internal standard DNA (log C)in the sample is obtained based on the measured fluorescence intensityand the amounts of internal standard DNA used in the chambers 104. Theregression curve can then be used to estimate the amount of target DNAin the sample. Quantitative analysis of the target DNA can be performedin this manner both accurately and efficiently.

The present invention encompasses configurations essentially the same asthose described in the embodiment (for example, a configuration with thesame functions, methods, and results, and a configuration with the sameobject and results). The invention also encompasses configurations thathave replaced non-essential components of the configurations describedin the embodiment. The invention also encompasses configurations thathave the same effects or achieve the same object as the configurationsdescribed in the embodiment. The invention also encompassesconfigurations that include the configurations of the foregoingembodiment in combination with the related art.

1. A biochip used for quantitative analysis of a target DNA contained ina sample, comprising: a type I chamber including; a primer that isdesigned to bind to the target DNA; a internal standard DNA of a firstamount that has a sequence different from a sequence of the target DNA,and is amplifiable with the primer; and a fluorescent probe that isdesigned to bind to a part of a PCR product of the target DNA and to apart of a PCR product of the internal standard DNA, and fluorescesdifferently for the PCR product of the target DNA and the PCR product ofthe internal standard DNA, and a type II chamber including; the internalstandard DNA of a second amount different from the first amount; and theprimer; and the fluorescent probe.
 2. The biochip according to claim 1,further comprising; a first path connected to the type I chamber and thetype II chamber; and a reservoir connected to the first path.
 3. Thebiochip according to claim 1, further comprising: a first group ofchambers including; the type I chamber and the type II chamber thatinclude a first primer used to amplify a first target DNA, and a secondgroup of chambers including; the type I chamber and the type II chamberthat include a second primer different from the first primer and used toamplify a second target DNA different from the first target DNA.
 4. Aquantitative method for a target DNA contained in a biological sampleusing a biochip, comprising: introducing a sample into a type I chamberand a type II chamber of the biochip; the type I chamber including; aprimer that is designed to bind to the target DNA; a internal standardDNA of a first amount that has a sequence different from a sequence ofthe target DNA, and is amplifiable with the primer; and a fluorescentprobe that is designed to bind to a part of a PCR product of the targetDNA and to apart of a PCR product of the internal standard DNA, andfluoresces differently for the PCR product of the target DNA and the PCRproduct of the internal standard DNA, and the type II chamber including;the internal standard DNA of a second amount different from the firstamount; the primer; and the fluorescent probe, performing a PCRamplification in the type I chamber and the type II chamber; measuring afluorescence intensity of the fluorescent probe in the type I chamberand the type II chamber; determining a regression curve of equation (1)below based on the measured fluorescence intensities in the type Ichamber and the type II chamber, and amounts of the internal standardDNA contained in the type I chamber and the type II chamber,F=a/(C+b)+c  (1) where F is a fluorescence intensity, C is an amount ofinternal standard DNA, b is an amount of target DNA contained in thesample, and a and c are predetermined values; and estimating the amountof the target DNA contained in the sample based on the regression curve.5. The method according to claim 4, wherein the regression curve isdetermined based on a relationship between a ratio of fluorescenceintensities that is measured before the PCR amplification and after thePCR amplification, and the amount of internal standard DNA used in thetype I chamber and the type II chamber.
 6. The method according to claim4, wherein the regression curve is determined based on a relationshipbetween (I) a ratio of a fluorescence intensity measured in a firststate in which the fluorescent probe binds to a part of the PCR productafter the PCR amplification, and a fluorescence intensity measured in asecond state in which the fluorescent probe dissociates from the PCRproduct after the PCR amplification, and (II) the amount of internalstandard DNA used in the type I chamber and the type II chamber.